Coenzyme-binding glucose dehydrogenase

ABSTRACT

The present invention provides a microorganism-derived soluble coenzyme-binding glucose dehydrogenase which catalyzes a reaction for oxidizing glucose in the presence of an electron acceptor, has an activity to maltose as low as 5% or less, and is inhibited by 1,10-phenanthroline. The invention also provides a method for producing the coenzyme-binding glucose dehydrogenase, and a method and a reagent for measuring employing the coenzyme-binding glucose dehydrogenase. According to the invention, the coenzyme-binding glucose dehydrogenase can be applied to an industrial field, and a use becomes possible also in a material production or analysis including a method for measuring or eliminating glucose in a sample using the coenzyme-binding glucose dehydrogenase as well as a method for producing an organic compound. It became also possible to provide a glucose sensor capable of accurately measuring a blood sugar level. Therefore, it became possible to provide an enzyme having a high utility, such as an ability of being used for modifying a material in the fields of pharmaceuticals, clinical studies and food products.

TECHNICAL FIELD

The present invention relates to a novel soluble coenzyme-bindingglucose dehydrogenase, a method for producing the coenzyme-bindingglucose dehydrogenase, and a microorganism having an ability ofproducing the coenzyme-binding glucose dehydrogenase.

The invention also relates to a method for measuring glucose in a sampleemploying the coenzyme-binding glucose dehydrogenase, and a reagent anda reagent composition containing the coenzyme-binding glucosedehydrogenase. Moreover, the invention relates to utilization inproducing and analyzing a material such as a starting material,including a method for producing an organic compound.

The invention further relates to a biosensor capable of rapidly andconveniently quantifying a particular component in a sample at a highaccuracy. Practically, the invention relates to a glucose sensoremploying the coenzyme-binding glucose dehydrogenase.

BACKGROUND ART

Glucose is present in blood and utilized as an important marker fordiabetes. A method for measuring a glucose has conventionally been achemical method or an enzymatic method, and an enzymatic method isregarded generally to be excellent in view of the specificity and thesafety. Such an enzymatic method is, for example, be a measurement usinga glucose oxidase, glucose-6-phosphate dehydrogenase or anNAD(P)-dependent glucose dehydrogenase. However, the methods employingthe glucose oxidase and the glucose-6-phosphate dehydrogenase are notconvenient reaction systems since they employ a plural of enzymes. Themethods employing the glucose-6-phosphate dehydrogenase and theNAD(P)-dependent glucose dehydrogenase pose a complication due to therequirement of adding a coenzyme NAD(P) to the reaction systems.

Recently, various biosensors were proposed as modes for quantifying aparticular component in a sample conveniently without diluting orstirring a sample solution. For example, a biosensor was proposed inwhich an electrode system consisting of an action electrode, a counterelectrode and a reference electrode was formed by a screen printing onan insulating board. This electrode system and the like was in contactwith an enzymatic reaction layer formed thereon containing a hydrophilicpolymer, a redox enzyme and an electron acceptor.

The number of diabetes patient is increasing year by year, and a methodfor measuring a blood sugar and a means for controlling the blood sugarlevel are desired which can be utilized not only in a hospital but alsoat home and which is convenient. While a simple glucose sensor isemployed currently for measuring the blood sugar, it frequently employsa glucose oxidase which is highly suspected to give a measured valueinvolving an error due to a level of the residual oxygen. On the otherhand, a biosensor employing a glucose dehydrogenase which is dependenton a nicotinamide-based coenzyme exhibits a high background noise andinvolves a complicated reaction system due to the requirement of addinga coenzyme or an auxiliary enzyme separately, and it also suffers from adisadvantage due to the requirement of an expensive optical system uponmeasuring a chromogenic system.

As an enzyme which is not affected by the residual oxygen level andwhich can act on glucose in the absence of NAD(P), a glucosedehydrogenase whose coenzyme is pyrroloquinolinequinone is known, butthe pyrroloquinolinequinone problematically tends to dissociate from theenzyme. A glucose dehydrogenase whose coenzyme ispyrroloquinolinequinone disclosed in JP-A-2000-350588 andJP-A-2001-197888 has a disadvantageously low selectivity for glucose. Onthe other hand, a glucose dehydrogenase whose coenzyme is thepyrroloquinolinequinone derived from Escherichia coli (JP-A-10-243786),a glucose dehydrogenase whose coenzyme is the pyrroloquinolinequinonederived from Pseudomonas sp. (Agric. Biol. Chem. (1980) 44:1505-1512)and a glucose dehydrogenase whose coenzyme is thepyrroloquinolinequinone derived from Gluconobacter suboxydans (Agric.Biol. Chem. (1981) 45:851-861) had the respective activity on maltose of3%, 3.2% and 5%, but were accompanied with the complication due to therequirement of a solubilization and an extraction of the enzymes sincethey were existing in a membrane fraction of the bacterial bodies.

The coenzyme-binding glucose dehydrogenases which oxidize a hydroxylgroup in the 3rd-position of glucose were also reported in J. Biol.Chem. (1967) 242: 3665-3672, Appl. Microbiol. Biotechnol. (1999) 51:58-64, Appl. Biochem. Biotechnol. (1996) 56: 301-310 and Enzyme Microb.Technol. (1998) 22:269-274, but any of them exhibits a poor selectivityfor glucose. Since maltose is employed widely as an infusion componentand the blood maltose level in an infused patient is high, it is desiredto develop an enzyme for measuring the blood sugar which is capable ofacting specifically on glucose and has low activity especially onmaltose.

In order to respond the industrial needs mentioned above, an objectiveof the invention is to provide a novel glucose dehydrogenase whichexhibits an excellent substrate-recognizing ability toward glucose andwhich has low activity on maltose, and also to provide a method forproducing the same and a microorganism having an ability of producingthe same.

Another objective of the invention is to provide excellent glucosemeasuring method, measuring reagent and biosensor which employ the novelglucose dehydrogenase and which are capable of quantifying glucoserapidly and conveniently at a high accuracy, as well as aglucose-eliminating reagent.

DISCLOSURE OF THE INVENTION

The invention is accomplished for solving the problems mentioned above,and the inventors made an effort in various ways and then focused on anovel soluble coenzyme-binding glucose dehydrogenase. Thecoenzyme-binding glucose dehydrogenase catalyzes a glucose-oxidizingreaction in the presence of an electron acceptor, and is classified forexample as EC (Enzyme Code) 1.1.99. The inventors also made an effort incharacterizing various microorganisms producing the coenzyme-bindingglucose dehydrogenase, and finally discovered a coenzyme-binding glucosedehydrogenase-producing microorganism and a coenzyme-binding glucosedehydrogenase.

The invention provides a glucose dehydrogenase to which a coenzyme isbound continuously upon a catalytic reaction. The coenzyme-bindingglucose dehydrogenase has a physicochemical ability of catalyzing areaction for oxidizing glucose, especially a hydroxyl group in the1st-position of glucose, in the presence of an electron acceptor. Thecoenzyme-binding glucose dehydrogenase has 5% or less activity tomaltose, preferably 3% or less; thus it has a poor activity on themaltose. On the other hand, the coenzyme-binding glucose dehydrogenaseallows its enzymatic activity to be inhibited characteristically by 50%or more at 5 mM of 1,10-phenanthroline, preferably by 50% or more at 2mM as a final concentration of 1,10-phenanthroline, more preferably by50% or more at 1 mM of 1,10-phenanthroline. The coenzyme-binding glucosedehydrogenase preserves its residual enzymatic activity at a level ashigh as 85% or more even after a heat treatment for 15 minutes at 50° C.in the presence of 50 mM sodium citrate buffer solution (pH5.5). Thecoenzyme which is bound to the inventive glucose dehydrogenase may forexample be a flavin compound, including a coenzyme such as flavinadenine dinucleotide. The invention also includes, with respect to aprotein having the characteristics of those of the coenzyme-bindingglucose dehydrogenase and/or characteristics equivalent substantiallythereto as well as its salt, a protein which has an amino acid sequenceencoding the protein or an amino acid sequence containing a mutationresulting from a deletion, substitution or addition of one or more aminoacid residues in the sequence and which is biologically active andstable. Moreover, the inventive coenzyme-binding glucose dehydrogenaseis a microorganism-derived coenzyme-binding glucose dehydrogenase,preferably a eukaryotic microorganism-derived coenzyme-binding glucosedehydrogenase, more preferably the deposited strain FERMBP-08578-derived coenzyme-binding glucose dehydrogenase.

It has already been observed that a glucose dehydrogenase whose coenzymeis a flavin adenine dinucleotide exists in a cytoplasm fraction and aculture of Aspergillus oryzae (TCHAN-GI BAK (BIOCHEMICA ET BIOPHYSICAACTA. (1967) 139:277-293)). However, this glucose dehydrogenase isinhibited only by a heavy metal ion, and characterized physicochemicallyin that it is not inhibited by a metal chelator including1,10-phenanthroline. Accordingly, in a measurement system employing thisglucose dehydrogenase, only a heavy metal can be used as a quencher,which poses a problem associated with a complicated heavy metal wastedisposal after completion of the reaction. In addition, this enzyme hasa poor stability, and is problematic when used practically. On the otherhand, the coenzyme-binding glucose dehydrogenase discovered in thisinvention is characterized by its higher stability when compared withthat of the known Aspergillus oryzae-derived coenzyme-binding glucosedehydrogenase, and also by a favorably convenient handling for quenchingbecause of its ability of being inhibited by a trace amount of1,10-phenanthroline in addition to a heavy metal ion.

The invention provides a method for producing the novel solublecoenzyme-binding glucose dehydrogenase.

The invention provides a microorganism having an ability of producingthe novel soluble coenzyme-binding glucose dehydrogenase. Themicroorganism is preferably a eukaryotic microorganism, more preferably,genus Aspergillus, further preferably, Aspergillus terreus, and mostpreferably, the deposited strain FERM BP-08578.

The invention provides a method using the novel soluble coenzyme-bindingglucose dehydrogenase. Preferably, a method for measuring glucose usingthe coenzyme-binding glucose dehydrogenase is provided for measuringglucose in a sample. A method for eliminating glucose using thecoenzyme-binding glucose dehydrogenase and a method for producing anorganic compound are also provided.

The invention provides a reagent containing the novel solublecoenzyme-binding glucose dehydrogenase. The reagent is preferably aglucose-measuring reagent containing the coenzyme-binding glucosedehydrogenase employed for measuring the glucose in a sample, and aglucose-eliminating reagent containing the coenzyme-binding glucosedehydrogenase as well as an organic compound-producing reagent.

The invention provides a reagent composition containing the novelsoluble coenzyme-binding glucose dehydrogenase. The composition ispreferably a glucose-measuring composition containing thecoenzyme-binding glucose dehydrogenase employed for measuring glucose ina sample, and a glucose-eliminating composition containing thecoenzyme-binding glucose dehydrogenase as well as an organiccompound-producing composition.

The invention provides a biosensor employing the novel solublecoenzyme-binding glucose dehydrogenase and a biosensor capable ofquantifying and/or qualifying a particular component in a sample. Such abiosensor is preferably a glucose sensor employing the coenzyme-bindingglucose dehydrogenase.

One preferred embodiment of these inventive measurement methods,measurement reagents, measurement compounds and biosensors ischaracterized by the use of potassium ferricyanide (potassiumhexacyanoferrate (III)) at a final concentration of 2 mM to 500 mM.

In the invention, a value of percentage (%) represents “substratespecificity”. For example, in the expressions “activity to maltose”,“activity” toward maltose” or an analogous expression, according to thecoenzyme-binding glucose dehydrogenase, such a value also represents fora percentage of a relative intensity of an enzymatic activity on themaltose or other action targets based on the enzymatic activity onglucose being regarded as 100%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph representing the relationship between the relativeactivity (%) of the coenzyme-binding glucose dehydrogenase and the pH,in Example 3 (3.2). Symbols show measured values and types of buffersolutions, which are □: citric acid-sodium phosphate buffer (pH4.6 to6.2), ♦: potassium phosphate buffer (pH6.8 to 7.2), ◯: Tris-HCl buffer(pH7.8 to 8.6) and ▴: glycine-sodium hydroxide (pH9.2 to 9.5). Theoptimum pH of the enzyme was 7.0 to 9.0.

FIG. 2 shows a graph representing the relationship between the residualactivity (%) of the coenzyme-binding glucose dehydrogenase and the pH,in Example 3 (3.3). Symbols show measured values and types of buffersolutions, which are □: citric acid-sodium phosphate buffer (pH3.2 to6.4), ♦: potassium phosphate buffer (pH6.3 to 6.9), ◯: Tris-HCl buffer(pH7.3 to 8.6) and ▴: glycine-sodium hydroxide (pH9.1 to 11.4). Thestable pH of the enzyme was 4.5 to 8.5.

FIG. 3 shows a graph representing the relationship between the relativeactivity (%) of the coenzyme-binding glucose dehydrogenase and thetemperature, in Example, 3 (3.4). The optimum temperature of the enzymewas approximately 55° C.

FIG. 4 shows a graph representing the relationship between the residualactivity (%) of the coenzyme-binding glucose dehydrogenase in Example 3(3.5) and the treatment temperature. The enzyme was revealed to bestable at 50° C. or below.

FIG. 5 shows a calibration curve for quantifying the glucose in Example4, which indicates the change in DCIP absorption vs the glucose level.

FIG. 6 shows a calibration curve for a glucose measurement using acoenzyme-binding glucose dehydrogenase-immobilizing electrode in Example5. Symbols represent the conditions under: x: argon saturation, Δ:oxygen saturation and ◯: air saturation.

BEST MODE FOR CARRYING OUT THE INVENTION

An inventive coenzyme-binding glucose dehydrogenase may for example bean enzyme classified as EC 1.1.99, preferably EC 1.1.99.10, EC 1.1.99.13or EC 1.1.99.17, and is a coenzyme-binding enzyme, preferably a solublecoenzyme-binding enzyme. That is, it is an enzyme capable of beingobtained in the state of an aqueous solution without using anysurfactant in a step of extraction/purification of the enzyme. As usedherein, a coenzyme may be any flavin compound, such as flavin adeninedinucleotide, flavin mononucleotide and the like.

The inventive coenzyme-binding glucose dehydrogenase may be a glucosedehydrogenase to which a coenzyme is bound always over the period of thecatalytic reaction. The coenzyme-binding glucose dehydrogenase has atleast the following characteristics; that is a physicochemical propertyfor catalyzing a reaction for oxidizing glucose in the presence of anelectron acceptor, especially for catalyzing a reaction for oxidizing ahydroxyl group in the 1st-position of the glucose. In addition, thecoenzyme-binding glucose dehydrogenase has a low activity on maltose,which is at a level for example of 5% or below, preferably 3% or below.Moreover, the enzymatic activity of the coenzyme-binding glucosedehydrogenase is inhibited by 50% or more at 5 mM of1,10-phenanthroline, preferably by 50% or more at 2 mM as a finalconcentration of 1,10-phenanthroline, more preferably by 50% or more at1 mM of 1,10-phenanthroline, especially by 60% or more at 1 mM of1,10-phenanthroline. The coenzyme-binding glucose dehydrogenasepreserves its residual enzymatic activity at a level as high as 85% ormore even after a heat treatment for 15 minutes at 50° C. in thepresence of 50 mM sodium citrate buffer solution (pH5.5). Moreover, theinvention relates to coenzyme-binding glucose dehydrogenase having thephysicochemical characteristics described above; or an amino acidsequence encoding the physicochemical characteristics, wherein the aminoacid sequence is a protein or a salt as follows: the amino acid sequenceencoding the protein or the salt contains a mutation resulting from adeletion, substitution or addition of one or more amino acid residues inthe sequence, and such the resultant protein and the salt arebiologically active and stable, with respect to the protein or its salthaving the activity substantially equivalent to that of thecoenzyme-binding glucose dehydrogenase.

The protein or its salt, which are the inventive coenzyme-bindingglucose dehydrogenase, is preferably one derived from a microorganismhaving the physicochemical characteristics described above. Amicroorganism from which the inventive coenzyme-binding glucosedehydrogenase is derived and a microorganism having an ability ofproducing the inventive coenzyme-binding glucose dehydrogenase may forexample be those classified into prokaryotic microorganisms such asgenus Archangium, genus Arcaeoglobus, genus Arsenophonus, genusAhrensia, genus Aureobacterium, genus Aerococcus, genus Aeropyrum, genusAeromicrobium, genus Aeromonas, genus Aquifex, genus Aquaspirillum,genus Aquabacter, genus Aquabacterium, genus Aquamicrobium, genusActinolloteichus, genus Actinokineospora, genus Actinocorallia, genusActinosynnema, genus Actinosporangium, genus Actinobaculum, genusActinobacilus, genus Actinopycnidium, genus Actinobispora, genusActinoplanes, genus Actinopolyspora, genus Actinopolymorpha, genusActinomyces, genus Actinomadura, genus Acrocarpospora, genus Agrococcus,genus Agrobacterium, genus Agromyces, genus Agromatium, genus Agromonas,genus Achromobacter, genus Acholeplasma, genus Asaia, genus Acidianus,genus Acidispaera, genus Acidiphilium, genus Acidimicrobium, genusAcidilobus, genus Acidaminococcus, genus Acidaminobacter, genusAcidithiobacillus, genus Agitococcus, genus Acidothermus, genusAcidocella, genus Acidobacterium, genus Acidovorax, genus Acidomonas,genus Acinetobacter, genus Asiticcacaulis, genus Asteroleplasma, genusAcetitomaculum, genus Acetivibrio, genus Acetoanaerobium, genusAcetogenium, genus Acetothermus, genus Acetonema, genus Acetobacter,genus Acetobacterium, genus Acetohalobium, genus Acetofilamentum, genusAcetomicrobium, genus Azoarcus, genus Azospira, genus Azospirillum,genus Azotobacter, genus Azonexus, genus Azovibrio, genus Azomonas,genus Azomonotrichon, genus Azorhizobium, genus Azorhizophilus, genusAtopobacter, genus Atopobium, genus Anaplasma, genus Aneurinibacillus,genus Anaeroarcus, genus Anaerococcus, genus Anaerosinus, genusAnaerobacter, genus Anaerobaculum, genus Anaerobispirillum, genusAnaerovibrio, genus Anaerofilum, genus Anaeroplasma, genus Anaerobranca,genus Anaerovorax, genus Anaeromusa, genus Anaerorhabdus, genusAnoxybacillus, genus Abiotrophia, genus Afipia, genus Amaricoccus, genusAmycolata, genus Amycolatopsis, genus Aminobacter, genus Aminobacterium,genus Aminomonas, genus Amoebobacter, genus Ammoniphilus, genusAmmonifex, genus Amorphosporangium, genus Arachnia, genus Alysiella,genus Alicyclobacillus, genus Alishewanella, genus Alcanivorax, genusArcanobacterium, genus Alcaligenes, genus Alkalibacterium, genusAlkaliphilus, genus Arcobacter, genus Arthrobacter, genus Alterococcus,genus Alteromonas, genus Albibacter, genus Alloiococcus, genusAllochromatium, genus Arhodomonas, genus Allomonas, genus Allorhizobium,genus Anacalochloris, genus Anacalomicrobium, genus Angiococcus, genusAngulomicrobium, genus Ancylobacter, genus Antarctobacter, genusAmphibacillus, genus Ampullariella, genus Ignavigranum, genusIdiomarina, genus Isochromatium, genus Isosphaera, genus Ideonella,genus Ilyobacter, genus Intrasporagium, genus Weeksella, genusWigglesworthia, genus Williamsia, genus Wolinella, genus wolbachia,genus Ureplasma, genus Ureibacillus, Eikenella, genus Ehrlichia, genusExiguobacterium, genus Excellospora, genus Ectothiorhodospira, genusAegyptianella, genus Eggerthella, genus Escherichia, genus Edwardsiella,genus Ewingella, genus Eperythrozonn, genus Erysipelothrix, genusErythrobacter, genus Erythromicrobium, genus Erythromonas, genusElytrosporangium, genus Yersinia, genus Erwinia, genus Eremococcus,genus Ensifer, genus Enterococcus, genus Enterobacter, genusEntomoplasma, genus Enhydrobacter, genus Empedobacter, Oenococcus, genusOerskovia, genus Oceanimonas, genus Oceanospirillum, genus Oxalobacater,genus Oxalophagus, genus Oxobacter, genus Octadecabacter, genusOchrobactrum, genus Oscillochloris, genus Oscillospira, genusObesumbacterium, genus Orientia, genus Oligella, genus Oligotropha,genus Oribaculum, genus Ornithinicoccus, genus Ornithinimicrobium, genusOrnithobacterium, genus Orenia, genus Gardnerella, genus Carnimonas,genus Carnobacterium, genus Couchioplanes, genus Cowdria, genusCaulobacter, genus Caseobacter, genus Catenibacterium, genusCatenuloplanes, genus Catenococcus, genus Catellatospora, genusCatonella, genus Capsularis, genus Capnocytophaga, genus Gallionella,genus Carvophanon, genus Gallicola, genus Calymmatobacterium, genusCardiobacterium, genus Caldicellulosiruptor, genus Caldivirga, genusCalderobacterium, genus Carboxydibrachium, genus Carboxydothermus, genusCarbophilus, genus Caloramator, genus Xanthobacter, Xanthomonas, genusXylella, genus Xylophilus, genus Xenorhabdus, genus Kitasatoa, genusKirasatospora, genus Chitinophaga, genus Kytococcus, genus Kineococcus,genus Kineosporia, genus Quinella, genus Kibdelosporangium, genusCampylobacter, genus Kingella, genus Kutzneria, genus Cupriavidus, genusCraurococcus, genus Glaciecola, genus Gracilibacillus, genusGranulicatella, genus Grahamella, genus Clavibacter, genus Chlamydia,genus Chlamydophila, genus Cryobacterium, genus Glycomyces, genusChrysiogenes, genus Cristispira, genus Chryseobacterium, genusChryseomonas, genus Crinalium, genus Cryptosporangium, genusCryprobacterium, genus Kluyvera, genus Kribbella, genusGluconacetobacter, genus Gluconobacter, genus Kurthia, genusCurtobacterium, genus Crenothrix, genus Klebsiella, genus Clevelandina,genus Crossiella, genus Clostridium, genus Clobicatella, genusCHromatium, genus Chromobacterium, genus Chromohalobacter, genusChloronema, genus Chrorobium, genus Chloroflexus, genus Chloroherpeton,genus Caedibacter, genus Ketogulonicigenium, genus Gemmata, genusGemmiger, genus Gemella, genus Gemmobacter, genus Chelatococcus, genusChelatobacter, genus Gelidibacter, genus Coenonia, genus Coxiella, genusKocuria, genus Koserella, genus Coprococcus, genus Coprothermobacter,genus Coprobacillus, genus Comamonas, genus Coriobacterium, genusCorynebacterium, genus Collinsella, genus Colwellia, genus Gordonia,genus Conglomeromonas, genus Chondromyces, genus Thermaerobacter, genusThermus, genus Thermacetogenium, genus Thermanaerovibrio, genusThermicanus, genus Thermithiobacillus, genus Thermoactinomyces, genusThermoanaerobacter, genus Thermoanaerobacterium, genus Thermoanaerobium,genus Thermocladium, genus Thermocrispum, genus Thermocrinis, genusThermochromatium, genus Thermococcus, genus Thermosipho, genusThermosyntropha, genus Thermosphaera, genus Thermothrix, genusThermodesulfobacterium, genus Thermodesulfovibrio, genusThermodesulforhabdus, genus Thermoterrabacterium, genus Thermotoga,genus Thermonema, genus Thermohydrogenium, genus Thermobacteroides,genus Thermobacillus, genus Thermohalobacter, genus Thermobispora, genusThermobifida, genus Thermofilum, genus Thermobranchium, genusThermoplasma, genus Thermoproteus, genus Thermomicrobium, genusThermomonospora, genus Thermoleophilum, genus Cytophaga, genusZymobacter, genus Zymophilus, genus Zymomonas, genus Sagittula, genusSaccharococcus, genus Saccharothrix, genus Saccharobacter, genusSaccharopolyspora, genus Saccharomonospora, genus Zavarzinia, genusSubtercola, genus Saprospira, genus Samsonia, genus Salinicoccus, genusSalinivibrio, genus Salibacillus, genus Sarcobium, genus Sarcina, genusSalmonella, genus Salegentibacter, genus Sanguibacter, genusSandaracinobater, genus Dyadobacter, genus Dialister, genus Dietzia,genus Shewanella, genus Geothrix, genus Geodermatophilus, genus Geotoga,genus Geobacter, genus Geobacillus, genus Geovibrio, genus Dictyoglomus,genus Cycloclasticus, genus Psychroserpens, genus Psychrobacter, genusCyclobacterium, genus Psychroflexus, genus Psychromonas, genus Sigella,genus Dichelobacter, genus Dichoromicrobium, genus Dysgonomonas, genusCystobacter, genus Citrobacter, genus Synergistes, genus Sinorhizobium,genus Diplocalyx, genus Simkania, genus Simonsiella, genus Janibacter,genus Janthinobacterium, genus Pseudaminobacter, genus Pseudoamyclata,genus Pseudoalteromonas, genus Pseudoxanthomonas, genusPseudocaedibacter, genus Pseudonocardia, genus Pseudobutyrivibrio, genusPseudomonas, genus Pseudoramibacter, genus Schwartzia, genus Jonesia,genus Johnsonella, genus Silicibacter, genus Syntrophus, genusSyntrophococcus, genus Syntrophothermus, genus Syntrophospora, genusSyntrophobacter, genus Syntrophobotulus, genus Syntrophomonas, genusSymbiotes, genus Symbiobacterium, genus Zoogloea, genus Duganella, genusSchineria, genus Succiniclasticum, genus Succinispira, genusSuccinivibrio, genus Succinimonas, genus Skermania, genus Skermanella,genus Starkeya, genus Stappia, genus Staphylococcus, genusStaphylothermus, genus Staleya, genus Stygiolobus, genus Stibiobacter,genus Stigmatella, genus Stetteria, genus Stenotrophomonas, genusStella, genus Suttrella, genus Suttonella, genus Stomatococcus, genusStreptoalloteichus, genus Streptococcus, genus Streptosporangium, genusStreptoverticillium, genus Streptobacillus, genus Streptomyces, genusStreptomonospora, genus Spirillum, genus Spirilliplanes, genusSpirillospora, genus Spirosoma, genus Spiroplasma, genus Spirochaeta,genus Sphingobacterium, genus Sphingobium, genus Sphingopyxis, genusSphaerotilus, genus Spaerobacter, genus Sphingomonas, genus Sporichthya,genus Sporocytophaga, genus Sporosarcina, genus Sporotomaculum, genusSporobacter, genus Sporobacterium, genus Sporohalobacter, genusSporomusa, genus Sporolactobacillus, genus Smithella, genus Slackia,genus Sulfitobacter, genus Sulfobacillus, genus Sulfophobococcus, genusSulfolobus, genus Sulfurisphaera, genus Sulfurococcus, genusSulforospirillum, genus Cedecea, genus Setobacterium, genus Sebaldella,genus Serratia, genus Seliberia, genus Cellvibrio, genus Cerpula, genusSerpulina, genus Serpens, genus Cellulosimicrobium, genus Cellulophaga,genus Cellulomonas, genus Selenihalanaerobacter, genus Selenomonas,genus Centipeda, genus Sodalis, genus Zobellia, genus Solobacterium,genus Thauera, genus Dactylosporangium, genus Tatumella, genusTatlockia, genus Thalassomonas, genus Thialkalicoccus, genusThialkalivibrio, genus Thialkalimicrobium, genus Thiocapsa, genusThiococcus, genus Thiodictyon, genus Thiocystis, genus Thiospira, genusThiospirillum, genus Thiosphaera, genus Thiothrix, genus Thiobacterium,genus THiobacillus, genus Thiohalocapsa, genus Thioflavicoccus, genusThiovulum, genus THioploca, genus THiopedia, genus Thiomargarita, genusThiomicrospira, genus Thiomonas, genus Thiolamprovum, genusThiorhodococcus, genus THiorhodospira, genus Thiorhodovibrio, genusTissierella, genus Chania, genus Tindallia, genus Tsukamurella, genusTuricella, genus Deinococcus, genus Deinobacter, genus Taylorella, genusTectibacter, genus Dechlorosoma, genus Dechloromonas, genusTessaracoccus, genus Desulfacinum, genus Desulfitobacterium, genusDesulfocapsa, genus Desulfococcus, genus Desulfosarcina, genusDesulfospira, genus Desulfosporosinus, genus Desulfocella, genusDesulfotalea, genus Desulfotignum, genus Desulfotomaculum, genusDesulfonatronum, genus Desulfonatronovibrio, genus Desulfonispora, genusDesulfonema, genus Desulfovirga, genus Desufobacter, genusDesulfobacterium, genus Desulfobacula, genus Desulfobacca, genusDesulfobulbus, genus Desulfohalobium, genus Desulfovibrio, genusDesulfofustis, genus Desulfofaba, genus Desulfofrigus, genusDesulfomicrobium, genus Desulfomonas, genus Desulfomonile, genusDesulforhabdus, genus Desulforhopalus, genus Desulfurella, genusDesulfurococcus, genus Desulfurobacterium, genus Desulfuromusa, genusDesulfuromonas, genus Desulfurolobus, genus Desemzia, genusDethiosulfovibrio, genus Tetragenococcus, genus Tetrasphaera, genusDenitrobacterium, genus Denitrovibrio, genus Dehalobacter, genusTepidimonas, genus Deferribacter, genus Defluvibacter, genus Devosia,genus Demetria, genus Terracoccus, genus Terrabacter, genus Derxia,genus Delftia, genus Dermacoccus, genus Dermatophilus, genusDermabacter, genus Telluria, genus Deleya, genus Dendrosporobacter,genus Toxothrix, genus Trabulsiella, genus Trichlorobacter, genustrichococcus, genus Tlumonas, genus Treponema, genus Dolosigranulum,genus Dolosicoccus, genus Tropheryma, genus Neisseria, genus Natrialba,genus Natrinema, genus Natroniella, genus Natronincola, genusNatronococcus, genus Natronobacterium, genus Natronomonas, genusNAtronorubrum, genus Nannocystis, genus Nitrococcus, genus Nitrospina,genus Nitrospira, genus Nitrosococcus, genus Nitrosospira, genusNitrosomonas, genus Nitorosolobus, genus Nitrobacter, genusNeochlamydia, genus Neorickettsia, genus Nesterenkonia, genus Nevskia,genus Neptunomonas, genus Nocardia, genus Nocardioides, genusNocardiopsis, genus Nonomuraea, genus Novosphingobium, genusVirgibacillus, genus Hydrogenobacter, genus Hydrogenovibrio, genusHydrogenophaga, genus Hydrogenophilus, genus Hyperthermus, genusHyphomicrobium, genus Hyphomonas, genus Paucimonas, genus Bacterionema,genus Bacteriovorax, genus Bacteroides, genus Bactoderma, genusVagococcus, genus Pasteuria, genus Pasteurella, genus Bacillus, genusPapillibacter, genus Hafnia, genus Hahella, genus Paracraurococcus,genus Parachlamydia, genus Paracoccus, genus Halanaerobacter, genusHalanaerobium, genus Paralactobacillus, genus Variovorax, genusHaliscomenobacter, genus Bartonella, genus Balneatrix, genusPalaeococcus, genus Hallella, genus Haloarcula, genus Haloincola, genusHalochromatium, genus Halococcus, genus Halothermothrix, genusHalogeometricum, genus Halospirulina, genus Halocella, genusHalothiobacillus, genus Haloterrigena, genus Halonatronum, genusHalobaculum, genus Halobacterium, genus Halobacteroides, genusHalobacillus, genus Halobvibrio, genus Haloferax, genusHalomethanococcus, genus Halomonas, genus Halorhabdus, genus Halorubrum,genus Halorubrobacterium, genus Halorhodospira, genus Pantoea, genusPandoraea, genus Vampirovibrio, genus Picrophilus, genusPiscirickettsia, genus Hippea, genus Vitreoscilla, genusBifidobacterium, genus Vibrio, genus Hymenobacter, genus Pimelobacter,genus Pilimelia, genus Hirschia, genus Pirella, genus Pirellula, genusPyrococcus, genus Pyrodictium, genus Pillotina, genus Pyrobaculum, genusBilophila, genus Pyrolobus, genus Faenia, genus Facklamia, genusPhascolarctobacterium, genus Falcivibrio, genus Fundibacter, genusFinegoldia, genus Fibrobacter, genus Filibacter, genus Filifactor, genusPhyllobacterium, genus Filobacillus, genus Filomicrobium, genusPhaeospirillum, genus Phenylobacterium, genus Ferriacterium, genusFerrimonas, genus Fervidobacterium, genus Ferroglobus, genusFerroplasma, genus Phocoenobacter, genus Photobactrium, genusPhotorhabdus, genus Formivibrio, genus Fusobacterium, genusButtiauxella, genus Butyrivibrio, genus Bdellovibrio, genus Budvicia,genus Phennigia, genus Buchnera, genus Fusibacter, genus Prauserella,genus Pragia, genus Bracyspira, genus Bracybacterium, genus Bracymonas,genus Blastochloris, genus Blastococcus, genus Blastobacter, genusBlastomonas, genus Blattabacterium, genus Bradyrhizobium, genusFrateuria, genus Branhamella, genus Planococcus, genus Planotetraspora,genus Planobispora, genus Planopolyspora, genus Planomicrobium, genusPlanomonospora, genus Flavimonas, genus Flavobacterium, genusFlammeovirga, genus Frankia, genus Planctomyces, genus Francisella,genus Friedmanniella, genus Frigoribacterium, genus Fluoribacter, genusBurkholderia, genus Brucella, genus Bulleidia, genus Flexistipes, genusFlexithrix, genus Flexibacter, genus Flectobacillus, genus Plesiomonas,genus Brenneria, genus Brevundimonas, genus Brevinema, genusBrevibacterium, genus Brevibacillus, genus Prevotella, genusProchlorococcus, genus Prochlorothrix, genus Prochloron, genusBrochothrix, genus Prosthecochloris, genus Prosthecobacter, genusProsthecomicrobium, genus Proteus, genus Protomonas, genusPropionigenium, genus Propionispira, genus Propionispora, genusPropionibacter, genus Propionibacterium, genus Propionivibrio, genusPropioniferax, genus Providencia, genus Promicromonospora, genusProlinoborus, genus Beijejrinckia, genus Veillonella, genusBeutenbergia, genus Beggiatoa, genus Pectinatus, genus Pectobacterium,genus Pediococcus, genus Pedobacter, genus Pedomicrobium, genusPetrotoga, genus Paenibacillus, genus Beneckea, genus Peptococcus, genusPeptostreptococcus, genus Peptoniphilus, genus Haemobartonella, genusHaemophilus, genus Heliothrix, genus HEliobacterium, genusHeliobacillus, genus HEliophilum, genus Heliorestis, genus Helicobacter,genus Pelistega, genus Pelczaria, genus Bergeyella, genus Helcococcus,genus Verrucosispora, genus Verrucomicrobium, genus Persicobacter, genusHerbaspirillum, genus Herbidospora, genus Herpetosiphon, genusPelodictyon, genus Pelospora, genus Pelobacter, genus Vogesella, genusBogoriella, genus Bosea, genus Polaribacter, genus Polaromonas, genusHollandina, genus Polyangium, genus Polynucleobacter, genus Volcaniella,genus Bordetella, genus Holdemania, genus Porphyrobacter, genusPorphyromonas, genus Borrelia, genus Holospora, genus Holophaga, genusHongia, genus Meiothermus, genus Mycobacterium, genus Mycoplasma, genusMycoplana, genus Mycetocola, genus Myroides, genus Magnetospirillum,genus Macrococcus, genus Macromonas, genus Massilia, genus Maricaulis,genus Marichromatium, genus Marinococcus, genus Marinitoga, genusMarinilabilia, genus Marinospirillum, genus Marinobacter, genusMarinobacterium, genus Marinomonas, genus Marmoricola, genusMalonomonas, genus Mannheimia, genus Micavibrio, genus Myxococcus, genusMicroellobosporia, genus Micrococcus, genus Microcyclus, genusMicrocystis, genus Microscilla, genus Microsphaera, genusMicrotetraspora, genus Microvirgula, genus Microbacterium, genusMicrobulbifer, genus Microbispora, genus Micropruina, genusMicropolyspora, genus Micromonas, genus Micromonospora, genusMicrolunatus, genus Mitsuokella, genus Megasphaera, genus Megamonas,genus Mesophilobacter, genus Mesoplasma, genus Mesorhizobium, genusMethanimicrococcus, genus Methanocalculus, genus Methanoculleus, genusMethanogenium, genus Methanocorpusculum, genus Methanococcoides, genusMethanococcus, genus Methanothermus, genus Methanothermobacter, genusMethanosaeta, genus Methanosarcina, genus Methanospirillum, genusMethanosphaera, genus Methanothrix, genus Methanobacterium, genusMethanohalobium, genus Methanohalophilus, genus Methanopyrus, genusMethanofollis, genus Methanoplanus, genus Methanoburevibacter, genusMethanomicrobium, genus Methanolacinia, genus Methanolobus, genusMethallosphaera, genus Methylarcula, genus Methylocaldum, genusMethylococcus, genus Methylcarcina, genus Methylocystis, genusMethylosinus, genus Methysphaera, genus Methylcella, genus Methylbacter,genus Methylbacterium, genus Methylobacillus, genus Methylopila, genusMethylophaga, genus Methylpholus, genus Methylovorus, genusMethylmicrobium, genus Methylomonas, genus Methylorhabdus, genusMeniscus, genus Melittangium, genus Melissococcus, genus Moellerella,genus Moorella, genus Mogibacterium, genus Modestobacter, genusMobiluncus, genus Moraxella, genus Morganella, genus Mortiella, genusMorococcus, genus Eubacterium, genus lodobacter, genus Yokenella, genusRahnella, genus Raoultella, genus Lactococcus, genus Lactosphaera, genusLactobacillus, genus Lachnospira, genus Rathayibacter, genusRhabdochromatium, genus Labrys, genus Ralstonia, genus Rarobacter, genusLamprocystis, genus Lamprobacter, genus Lampropedia, genus Riemerella,genus Rickettsia, genus Rickettsiella, genus Rikenella, genus Listeria,genus Listonella, genus Lysobacter, genus Rhizobacter, genus Rhizobium,genus Rhizomonas, genus Lyticum, genus Limnobacter, genus Lewinella,genus Ruegeria, genus Rugamonas, genus Lucibacterium, genus Luteimonas,genus Luteococcus, genus Runella, genus Rubrivivax, genus Rubrimonas,genus Rubrobacter, genus Ruminococcus, genus Ruminobacter, genusLeifsonia, genus Leclercia, genus Lechevalieria, genus Legionella, genusRenibacterium, genus Levinea, genus Leptospira, genus Leptospirillum,genus Leptothrix, genus Leptotrichia, genus Leptonema, genusLeminorella, genus Lentzea, genus Leucothrix, genus Leuconostoc, genusLeucobacter, genus Lawsonia, genus Lautropia, genus Lochalimaea, genusRoseateles, genus Roseinatronobacter, genus Roseibium, genus Roseivivax,genus Roseococcus, genus Roseospira, genus Roseospirillum, genusRoseobacter, genus Roseovarius, genus Roseomonase, genus Roseburia,genus Rhodanobacter, genus Rothia, genus Rhodococcus, genusRhodothermus, genus Rhodocyclus, genus Rhodocista, genusRhodopseudomonas, genus Rhodospira, genus Rhodospirillum, genusRhodothalassium, genus Rhodobaca, genus Rhodobacter, genus Rhodobium,genus Rhodovibrio, genus Rhodopila, genus Rhodoferax, genus Rhodoplanes,genus Rhodovulum, genus Rhodomicrobium, genus Lonepinella, genusWeissella, genus Waddlia and the like.

Those which can also be exemplified are eukaryotic microorganisms suchas genus Issatchenkia, Candida, genus Cryptococcus, genus Kluyverintces,genus Kloeckera, genus Saccharomycodes, genus Saccharomyces, genusZygosaccharomyces, genus Shizosaccharomyces, genus Sirobasidium, genusStrigmatomyces, genus Sporidobolus, genus Sporobolomyces, genus Dekkera,genus Debaryomyces, genus Trichosporon, genus Trigonopsis, genusTorulaspora, genus Tremella, genus Nadsonia, genus Nematospora, genusHanseniaspora, genus Pichia, genus Fibulobasidium, genus Filobasidium,genus Filobasidiella, genus Bullera, genus Brettanomyces, genusHoltermannia, genus Malassezia, genus Metschnikowia, genus Lipomyces,genus Leucosporidium, genus Rhodosporidium, genus Rhodotorula, genusAcaulopage, genus Aquamortierella, genus Asellaria, genus Amoebidium,genus Amoeophilus, genus Arundinula, genus Utharomyces, genusEchinosporangium, genus Enterobryus, genus Endogone, genusEntomophthora, genus Kickxella, genus Genistellospora, genusChoanephora, genus Coemansia, genus Cochlonema, genus Conidiobolus,genus Saksenaea, genus Thamnidium, genus Thamnocephalis, genus Dispira,genus Dimargaris, genus Syncephalastrum, genus Syncephalis, genusZoopage, genus Sclerocystis, genus Smittium, genus Basidiobolus, genusParataeniella, genus Paramoebidium, genus Palavascia, genus Harpella,genus Piptocephalis, genus Pilobolus, genus Phycomyces, genus Blakeslea,genus Hesseltinella, genus Helicocephalum, genus Mycotypha, genusRadiomyces, genus Legeriomyces, genus Rhopalomyces, genus Acrasis, genusAcytostelium, genus Arcyria, genus Echinostelium, genusEchinosteliopsis, genus Oligonema, genus Cavostelium, genusGuttulinopsis, genus Clastoderma, genus Cribraria, genus Coenonia, genusCopromyxa, genus Comatricha, genus Colloderma, genus Dianema, genusDictyostelium, genus Didymium, genus Diderma, genus Stemonitis, genusThraustochytrium, genus Ceratiomyxa, genus Ceratiomyxella, genusTrichia, genus Physarum, genus Plasmodiophrora, genus Fuligo, genusBursulla, genus Prorostelium, genus Protosporangium, genus Hemitrichia,genus Perichaena, genus Polysphondylium, genus Polymyxa, genusLabyrinthula, genus Lamproderma, genus Lycogala, genus Licea, genusWardmyces, genus Actinopelte, genus Asterosporium, genus Arthrinium,genus Alternaria, genus Oidium, genus Clabosporium, genus Cladobotryum,genus Graphium, genus Colletotrichum, genus Sclerotium, genusStagonospora, genus Stibella, genus Tubercularia, genus Bactridium,genus Pycnothrium, genus Phaeoisaria, genus Pestalozziella, genusRhizoctonia, genus Rhinocladiella, genus Leptothyrium, genusAchlyogeton, genus Anisolpidium, genus Albugo, genus Ectrogella, genusOlipidium, genus Olpidiopsis, genus Catenaria, genus Chytridium, genusCladochytrium, genus Coelomomyces, genus Gonapodya, genus Saprolegnia,genus Sirolpidium, genus Synchytrium, genus Haliphthoros, genusHarpochytrium, genus Pythium, genus Hyphochytrium, genus Physoderma,genus Phlyctidium, genus Blastocladia, genus Peronospora, genusPeronophythora, genus Micormycopsis, genus Megachytrium, genusMonoblepharis, genus Lagenidium, genus Rhizidiomyces, genus Rhizidium,genus Rhipidium, genus Leptomitus, genus Leptolegniella, genusAcremonium, genus Aspergillus, genus Absidia, genus Arachniotus, genusArthrobotrys, genus Ulocladium, genus Echinobotryum, genus Exophiala,genus Epicoccum, genus Oidiodendron, genus Oedocephalum, genusAureobasidium, genus Curvularia, genus Candelabrella, genusCunninghamella, genus Gymnoascus, genus Cladosporium, genus Graphium,genus Gliocladium, genus Chrysosporium, genus Chromelosporium, genusGeotrichum, genus Geomyces, genus Chaetomium, genus Geniculifera, genusGonatobotrysm, genus Coniothyrium, genus Circinella, genus Zygorhynchus,genus Diplodia, genus Cylindrocarpon, genus Scopulariopsis, genusStachybotrys, genus Stemphylium, genus Sporothrix, genus Sepedonium,genus Dactylella, genus Talaromyces, genus Dratomyces, genus Trichurus,genus Trichocladium, genus Trichothecium, genus Trichoderma, genusTrichophyton, genus Nigrospora, genus Verticicladiella, genusVerticillium, genus Paecilomyces, genus Pithomyces, genus Bipolaris,genus Pyrenochaeta, genus Phialocephala, genus Phialophora, genus Phoma,genus Fusarium, genus Pestalotiopsis, genus Penicillium, genus Botrytis,genus Microsporum, genus Myrothecium, genus Mucor, genus Memnoniella,genus Monacrosporium, genus Monilia, genus Mortierella, genusEupenicillium, genus Eurotium, genus Rhizopus, genus Leptographium,genus Robillarda, genus Austroboletus, genus Auricularia, genusAuriscalpium, genus Agaricus, genus Agrocybe, genus Asterophona, genusAstraeus, genus Aseroe, genus Anellaria, genus Amauroderma, genusAmanita, genus Armillaria, genus Armillariella, genus Alcuria, genusIschnoderma, genus Inocybe, genus Inonotus, genus Ileodictyon, genusWynnea, genus Verpa, genus Volvariella, genus Urnula, genusEchinodontium, genus Exidia, genus Elfvingia, genus Oudemansiella, genusOmphalina, genus Onnia, genus Catathelasma, genus Ganoderma, genusCamarophyllus, genus Chalciporus, genus Galerina, genus Calocera, genusCalostoma, genus Cantharellus, genus Cantharellula, genus Cyathus, genusCyclomyces, genus Cystoderma, genus Cyptotrama, genus Cymatoderma, genusGymnopilus, genus Kuehneromyces, genus Gyrodon, genus Gyroporus, genusGyromitra, genus Guepinia, genus Xanthoconium, genus Xylaria, genusXerocomus, genus Xeromphalina, genus Cudonia, genus Clavatia, genusClavaria, genus Clavariadelphus, genus Clavicorona, genus Clavulina,genus Clavulinopsis, genus Craterellus, genus Clathrus, genus Clitocybe,genus Clitopilus, genus Crinipellis, genus Grifola, genus Cryptoderma,genus Cryptoporus, genus Crucibulum, genus Creolophus, genus Crepidotus,genus Chroogomphus, genus Chlorosplenium, genus Geastrum, genusGeolossum, genus Cotylidia, genus Conocybe, genus Kobayashia, genusCoprinus, genus Gomphidius, genus Gomphus, genus Coriolus, genusCordyceps, genus Cortinarius, genus Coltricia, genus Collybia, genusSarcoscypha, genus Sarcodon, genus Sarcodontia, genus Suillus, genusSchizophyllum, genus Squamanita, genus Scutellinia, genus Scleroderma,genus Stereum, genus Strobilomyces, genus Stropharia, genus Spathularia,genus Sparassis, genus Daedaleopsis, genus Dacryomyces, genus Daldinia,genus Dictyophora, genus Tylopilus, genus Tyromyces, genus Descolea,genus Thelephora, genus Tulostoma, genus Trametes, genus Trichoglossum,genus Torichocoma, genus Tricoloma, genus Tricholomopsis, genusTremella, genus Tremellodon, genus Naematoloma, genus Nidula, genusNeobulgaria, genus Baeospora, genus Paxillus, genus Battarea, genusPanaeolus, genus Panus, genus Panellus, genus Bankera, genus Hygrocybe,genus Hygrophorus, genus Hygrophoropsis, genus Bisporella, genusPisolithus, genus Hydnum, genus Hydnellum, genus Hypsizygus, genusPiptoporus, genus Hypoxylon, genus Hymenochaete, genus Hirschioporus,genus Pyrrhoderma, genus Favolus, genus Phaeolus, genus Phaeolepiota,genus Phallus, genus Fistulina, genus Phyllotopsis, genus Phylloporus,genus Filoboletus, genus Phellinus, genus Fomitopsis, genus Fomes, genusPholiota, genus Psathyrella, genus Psilocybe, genus Pseudocolus, genusPseudohiatula, genus Prerula, genus Flammulina, genus Pulveroboletus,genus Bulgaria, genus Pluteus, genus Pleurocybella, genus Pleurotus,genus Plectania, genus Phlogiotis, genus Peziza, genus Penicilliopsis,genus Hebeloma, genus Hericium, genus Helvella, genus Podostroma, genusPolyozellus, genus Polyporus, genus Polyporellus, genus Holtermannia,genus Boibitius, genus Porphyrellus, genus Boletinus, genus Boletellus,genus Boletus, genus Boletopsis, genus Porodisculus, genus Bondarzewia,genus Macrocystidia, genus Macropodia, genus Macrolepiota, genusMarasmius, genus Marasmiellus, genus Microporus, genus Mycena, genusMitrula, genus Mutinus, genus Melanoleuca, genus Merulius, genusMorchella, genus Laetiporus, genus Lactarius, genus Lasiosphaera, genusLaccaria, genus Ramaria, genus Lampteromyces, genus Lyophyllum, genusRigidoporus, genus Lycoperdon, genus Rhizina, genus Lysurus, genusLimacella, genus Linder, genus Russula, genus Leucocoprinus, genusLeucopaxillus, genus Leotia, genus Resupinatus, genus Leccinum, genusLepiota, genus Lepista, genus Lenzites, genus Lentaria, genus Lentinus,genus Lentinula, genus Lentinellus, genus Rozites, genus Rhodocybe,genus Rhodotus, genus Rhodophyllus and the like.

A microorganism listed above which has an ability of producing aninventive coenzyme-binding glucose dehydrogenase has been depositedunder its accession number to IFO, ATCC and the like, and can beobtained from known distributors and corporations. Such a microorganismmay for example be a eukaryotic microorganism, more preferably mycoticmicroorganism. It is also possible to use the microorganism which isdesignated as “97508” and was deposited under FERM BP-08578 toInternational Patent Organism Depositary of National Institute ofAdvanced Industrial Science and Technology.

In one embodiment of the method for producing the inventivecoenzyme-binding glucose dehydrogenase, a microorganism having anability of producing the inventive coenzyme-binding glucosedehydrogenase is cultured in a nutrition medium, in which thecoenzyme-binding glucose dehydrogenase is allowed to be produced andaccumulated, and then recovered to yield a protein which is thecoenzyme-binding glucose dehydrogenase and its salt.

The deposited inventive strain FERM BP-08578, a coenzyme-binding glucosedehydrogenase derived therefrom, and a method for obtaining the enzymeare described below.

1. Physicochemical Characteristics of FERM BP-08578-Derived Enzyme

(1) Effect: On the basis of the classification by International Union ofBiochemistry (IUB), the inventive enzyme corresponds to EC1.1.99.10, andcatalyzes the reaction shown below which oxidizes a hydroxyl group inthe 1st-position of glucose in the presence of an electron acceptor toyield glucono-6-lactone.Glucose+Electron acceptor→Glucono-δ-lactone+reduced electron acceptor

In the invention, the electron acceptor may for example be phenazinemethosulfate, 1-methoxy-5-methylphenazinium methyl sulfate,2,6-dichlorophenolindophenol, ferricyanides and the like.

(2) Substrate specificity: The relative reactivity (substratespecificity) of the inventive enzyme when using D-glucose and othersubstrates (all at the final concentration of 333 mM except forD-cellobiose at 193 mM, D-trehalose and D-raffinose at 121 mM) by theactivity measurement method 1 described below are shown in Table 1. Therelative reactivity (substrate specificity) when using D-glucose andmaltose at the final concentrations of 550 mM and 100 mM are shown inTable 2. A higher activity was observed on D-glucose, while loweractivity were observed on D-mannose, 1,5-anhydro-D-glucitol,D-cellobiose, D-trehalose, maltose, D-galactose, D-glucose-6-phosphateand D-fructose. Almost no activity was observed on L-arabinose, lactose,D-sorbitol, gluconic acid, sucrose, D-mannitol, L-sorbose, D-ribose,L-rhamnose, D-glucose-1-phosphate, D-raffinose, ethanol or glycerol.(3) Optimum pH: pH7.0 to pH9.0.(4) pH for stability: pH4.5 to pH8.5.(5) Optimum temperature: Approximately 55° C.(6) Thermal stability: Stable at 50° C. or below.(7) Molecular weight: About 130 kDa when measured by a gel filtrationmethod, and about 85 kDa when measured by a sodium dodecylsulfate-polyacrylamide gel electrophoresis.(8) Km value: 49.7 mM (D-glucose).(9) Isoelectric point: The isoelectric point (pI) of thecoenzyme-binding glucose dehydrogenase measured by an isoelectricfocusing was about 4.4.(10) Inhibitor: When each additive was added at 1 mM as a finalconcentration to the activity measurement method 1 described below andthe activity was measured, an inhibitory effect of each additive wasobserved when comparing with the control group as shown in Table 3. Whenadding 1,10-phenanthroline (dissolved in methanol) at each finalconcentration to the activity measurement method 1 described below, theinhibitory effect shown in Table 4 was observed. The activity of thisenzyme was inhibited potently by heavy metal ions (Ag+, Cu²⁺, Hg²⁺), andinhibited by 60% or higher by 1,10-phenanthroline, proflavin and Mn²⁺.(11) Coenzyme: Flavin adenine dinucleotide.

The amino acid sequence of the coenzyme-binding glucose dehydrogenaseand the base sequence of a gene encoding it are also encompassed by theinvention.

For producing the inventive coenzyme-binding glucose dehydrogenase, amicroorganism for producing the coenzyme-binding glucose dehydrogenasemay be any microorganism as far as it can produce the inventivecoenzyme-binding glucose dehydrogenase, and the enzyme can efficientlybe produced by using a microorganism, preferably a eukaryoticmicroorganism, more preferably a mycotic microorganism. It is especiallypreferred to use the microorganism which is designated as “97508” andwas deposited under FERM BP-08578 to International Patent OrganismDepositary of National Institute of Advanced Industrial Science andTechnology. The deposited strain was isolated from a soil by Applicants,and has the mycological characteristics as described below. In theinvention, any variant of the strain mentioned above may also beemployed. Such a variant can be obtained for example by the irradiationwith an ultraviolet light or X-ray or the treatment with a chemicalmodifier (NTG and the like).

2. Mycological Characteristics of FERM BP-08578

(1) Morphological characteristics: The morphological profile of thepresent strain when observed by an optical microscope after allowed togrow on a potato dextrose agar medium is described below. Each myceliumhas a width of 2 μm to 4 μm, and has a regular septum. Most of themycelia grow linearly, and have branches with almost no swollen myceliabeing observed. Several mycelia are gathered together to form a mycelialbundle. The mycelial width is almost constant. The surface of a myceliumis smooth and the septum is slightly thick. A crystalloid is formedaround the root of the aerial mycelia. No clamp connection is formed.The mycotic body formed in 2-week culture exhibited no formation of anysexual or asexual reproductive organs, and no oidia or thick-walledspores are present.(2) Growth condition in various culture media: On all agar plates, eachmycelium is in a form of a fluff. The aerial mycelium shade is white. Onthe potato dextrose agar plate, the backside color is pale orange toorange. The growth magnitude is medium, and a colony after culturing at25° C. for 1 week has a diameter of 30 mm to 35 mm on the potatodextrose and oatmeal agar plates, and 37 mm to 38 mm on the malt extractagar plate. The production of soluble pigments of a pale yellow color onthe potato dextrose agar plate and a slightly reddish to grey-reddishcolor on the oatmeal agar plate was noted. The mycotic body formed in2-week culture exhibited no formation of reproductive organs such as aconidiophore, and no exudate was produced.(3) Physiological characteristics: The present strain is an aerobic oneand has an optimum growth temperature of about 37° C. on the potatodextrose agar plate.3. Taxonomical Characteristics of FERM BP-08578

Based on the characteristics described above, the deposited strain“97508” was characterized with referring to Ainsworth & Bisby'sDictionary of the Fungi, 7th edition (Ed. by Hawksworth, Sutton,Ainsworth). As a result of this characterization, the deposited strainwas revealed to be a microorganism classified into the genusAspergillus. Then its genome sequence was subjected to BLAST homologysearch. An 18S rDNA fragment was amplified using a genome DNA as atemplate by PCR method, and then sequence of the purified PCR productwas analyzed. In order to search for an analogous base sequence throughGenBank (GenBank/EMBL/DDBJ international DNA sequence database), BLAST(Altschul et al., 1997) homology search was conducted, and revealed thatthis deposited strain “97508” is Aspergillus terreus.

In one embodiment of the method for producing a coenzyme-binding glucosedehydrogenase according to the invention, the microorganism describedabove is cultured to allow the coenzyme-binding glucose dehydrogenase tobe expressed or produced by the microorganism in internal and/orexternal of its fungus body.

For the culture of the microorganism in the invention, any ordinaryculture medium for microorganism may be used. Such medium may include asynthetic or natural medium, as long as it contains suitable amounts ofcarbon sources, nitrogen sources, inorganic substances and other traceelements required by the microorganism. The carbon sources may beglucose, sucrose, dextrin, starch, glycerin, syrup and the like. Thenitrogen sources may be inorganic salts such as ammonium chloride,ammonium nitrate, ammonium sulfate and ammonium phosphate, amino acidssuch as DL-alanine and L-glutamic acid, as well as nitrogen-containingnatural materials such as peptone, meat extract, yeast extract, maltextract, corn steep liquor and the like. The inorganic materials may forexample be monosodium phosphate, disodium phosphate, monopottasiumphosphate, dipotassium phosphate, magnesium sulfate, ferric chloride andthe like.

The culture in order to obtain the inventive coenzyme-binding glucosedehydrogenase is conducted preferably in an aerobic condition forexample by a shaking or aerating culture at a temperature of 25° C. to60° C. at a pH of 5 to 8. The culture period ranges preferably from 2days to 4 days. As a result of such a culture, the coenzyme-bindingglucose dehydrogenase can be produced and accumulated in the culture,especially in a culture fluid. By using this culture method, thecoenzyme-binding glucose dehydrogenase can be produced by themicroorganism and accumulated also internal fungus body. Subsequently,the coenzyme-binding glucose dehydrogenase can be recovered from theculture by means of an ordinary protein purification method. Such amethod may be a method comprising incubating the microorganism followedby removing the microorganism for example by a centrifugation to obtaina supernatant, or a method comprising incubating the microorganism,recovering the cultured microorganism from the culture fluid by acentrifugation, crushing the cultured microorganism by a suitablemethod, and then isolating a supernatant for example by a centrifugationfrom the pelletized microorganism fluid. The coenzyme-binding glucosedehydrogenase contained in such a supernatant can be purified by acombination of suitable purification procedures such as salting out,solvent sedimentation, dialysis, ion exchange chromatography,hydrophobic adsorption chromatography, gel filtration, affinitychromatography, electrophoresis and the like.

In the culture for obtaining the inventive coenzyme-binding glucosedehydrogenase, a solid medium can be also used. The method for such aculture is not limited particularly, and may for example be a staticculture, or a dynamic culture involving a continuous agitation of theculture, such as a rotational culture or fluidized bed culture, with astatic culture being preferred due to a less expensive investment inequipment. Thereafter, as a method of obtaining the coenzyme-bindingglucose dehydrogenase from the culture, an ordinary protein purificationmethod can be adopted. That is, the culture is combined with water orother extraction medium and shaken, made free of any solid componentssuch as bran by means of a centrifugation or filtration, therebyyielding an extract. It is also possible that the coenzyme-bindingglucose dehydrogenase accumulated in the fungus bodies can be recoveredby grinding the culture residue after obtaining the extract describedabove together with a an abrasive compound such as a sea sand, followedby adding water to extract the coenzyme-binding glucose dehydrogenasereleased from the fungus bodies. In order to obtain the entirecoenzyme-binding glucose dehydrogenase, the entire culture is groundwith an abrasive compound such as a sea sand, followed by adding waterto extract both of the coenzyme-binding glucose dehydrogenase releasedfrom the fungus bodies and the coenzyme-binding glucose dehydrogenasesecreted into the culture all at once. The coenzyme-binding glucosedehydrogenase contained in such a supernatant can be purified by acombination of suitable purification procedures such as salting out,solvent sedimentation, dialysis, ion-exchange chromatography,hydrophobic adsorption chromatography, gel filtration, affinitychromatography, electrophoresis and the like.

Alternatively, the inventive coenzyme-binding glucose dehydrogenase mayalso be a synthetic coenzyme-binding glucose dehydrogenase or arecombinant coenzyme-binding glucose dehydrogenase obtained by a geneengineering technology. Those skilled in the art can obtain thecoenzyme-binding glucose dehydrogenase readily based on the disclosureof the protein or its salt derived from the physicochemicalcharacteristics of the inventive coenzyme-binding glucose dehydrogenase.For example, the coenzyme-binding glucose dehydrogenase can be extractedfrom a microorganism including a fungi or a naturally occurring materialsuch as an animal or plant, or can be obtained synthetically withreferring to the amino acid sequence or the base sequence of the geneencoding it. Moreover, it is also possible to produce thecoenzyme-binding glucose dehydrogenase industrially using a geneengineering method in which a gene segment of the coenzyme-bindingglucose dehydrogenase gene is inserted into a known expression vectorsuch as a commercial expression vector and then the resultant plasmid isused to transform a host such as Escherichia coli to obtain atransformant which is then cultured to obtain a target coenzyme-bindingglucose dehydrogenase.

For measuring the activity of the inventive enzyme, the enzyme isdiluted appropriately to a final concentration of 0.1 to 1 unit/mi. Theenzymatic activity unit of the enzyme is the enzymatic activity enablingthe oxidation of 1 μmol glucose per minute. The enzymatic activity ofthe inventive coenzyme-binding glucose dehydrogenase can be measured bythe method shown below.

(i) Enzymatic Activity Measurement Method 1

To a 3-ml quartz cell (light path length: 1 cm), 1.0 ml of 0.1Mpotassium phosphate buffer (pH7.0), 1.0 ml of 1.0M D-glucose, 0.1 ml of3 mM 2,6-dichlorophenolindophenol (hereinafter referred to as DCIP), 0.2ml of 3 mM 1-methoxy-5-methylphenazinium methyl sulfate and 0.65 ml ofwater are added, which is followed by setting in a thermostat cellholder-mounted spectrophotometer and incubated at 37° C. for 5 minutesand then supplemented with 0.05 ml of the enzyme solution, and thenmeasuring the change in the absorption of DCIP at 600 nm (AABS/min).Based on a molar extinction coefficient of DCIP at pH7.0 being regardedas 16.3×10³ cm⁻¹M⁻¹ and the enzymatic activity corresponding to thereduction of 1 μmol of DCIP per minute being equivalent to 1 unit of theenzymatic activity, the enzymatic activity was determined from thechange in the absorption on the basis of the following equation.Enzymatic activity (unit/ml)=(−ΔABS/16.3)×(3.0/0.05)×Enzyme dilutionrate(ii) Enzymatic Activity Measurement Method 2

It was carried out that 3.4 μl of 1.0M potassium phosphate buffer(pH7.0), 0.1 ml of 1.0M D-glucose and 86.6 μl of 20 mM DCIP wereincubated at 37° C. for 5 minutes, supplemented with 0.01 ml of anenzyme solution, stirred, reacted for 5 minutes, incubated at 100° C.for 3 minutes to quench the reaction. Thereafter, 0.19 ml of 100 mMglycine-sodium buffer (pH13.0), 0.01 ml of 2.0N potassium hydroxide wereadded, incubated at 37° C. for 10 minutes to convert D-gluconic acid inthe solution into D-glucono-6-lactone, and thereafter combined with 0.39ml of a 100 mM Tris-HCl buffer (pH7.5) and 0.01 ml of 1.0N hydrochloricacid to achieve a neutral pH. The D-gluconic acid in the solution wasquantified using the D-gluconic acid/D-glucono-6-lactone measurement kit(Boehringer Mannheim). Since the enzymatic activity corresponding to theproduction of 1 μmol of D-glucono-δ-lactone per minute is equivalentsubstantially to 1 unit of this enzyme, this enzymatic activity wasdetermined based on the amount of D-glucono-6-lactone produced.

The invention relates to a material production and an analyticalapplication employing an inventive coenzyme-binding glucosedehydrogenase, and also relates to the use in the modification ofpharmaceutical or food product materials. In one example, the use ismade in a method for eliminating glucose in a sample containing abiological material using the coenzyme-binding glucose dehydrogenase asa reagent, a measurement method as well as in such a reagent or areagent composition. The use is made also in a method for producing anorganic compound using the inventive coenzyme-binding glucosedehydrogenase as well as in a basic ingredient therefor.

The inventive coenzyme-binding glucose dehydrogenase is the enzyme thatcatalyzes a reaction for oxidizing glucose in the presence of anelectron acceptor. In the invention, the reaction mentioned aboveemploys the inventive coenzyme-binding glucose dehydrogenase. Such acoenzyme-binding glucose dehydrogenase is not limited particularly, andis preferably a coenzyme-binding glucose dehydrogenase derived from aeukaryotic microorganism producing the coenzyme-binding glucosedehydrogenase, with a coenzyme-binding glucose dehydrogenase derivedfrom a mycotic microorganism being preferred especially.

Now the description is made with regard to the application of thecoenzyme-binding glucose dehydrogenase obtained according to theinvention. Since the coenzyme-binding glucose dehydrogenase is theenzyme that catalyzes a reaction for oxidizing glucose in the presenceof an electron acceptor, any application in which the change resultingfrom such a reaction can be utilized can be mentioned. For example, thecoenzyme-binding glucose dehydrogenase can be used in a reagent formeasuring or eliminating glucose in a sample containing a biologicalmaterial. It is also possible to use in medical and clinical fields, andalso in the material production and analysis employing thecoenzyme-binding glucose dehydrogenase.

The inventive biosensor may be any sensor having a reaction layercontaining the inventive coenzyme-binding glucose dehydrogenase as anenzyme. For example, the biosensor can be produced in a method whereinan enzymatic reaction layer formed thereon containing a hydrophilicpolymer, a redox enzyme and an electron acceptor is brought into contactwith an electrode system consisting of an action electrode, a counterelectrode and a reference electrode is formed for example by a screenprinting on an insulating board. When a substrate-containing samplesolution is dropped onto the enzymatic reaction layer in this biosensor,the enzymatic reaction layer is dissolved to effect the reaction betweenthe enzyme and the substrate, thereby reducing the electron acceptor.After completion of the enzymatic reaction, the reduced electronacceptor is oxidized electrochemically, whereupon the biosensor canmeasure the concentration of the substrate in the sample based on theresultant oxidation current value. Otherwise, a biosensor can beconstructed so that the chromogenic intensity or pH change is detected.By using above mentioned biosensors, any of various materials can bemeasured by selecting the enzyme whose substrate is the targetsubstance. For example, when the inventive coenzyme-binding glucosedehydrogenase is selected as an enzyme, a glucose sensor enabling themeasurement of the concentration of glucose in a sample solution can beproduced.

As the electron acceptor in the biosensor, a chemical substance havingan excellent ability of transferring an electron can be employed. Such achemical substance having an excellent ability of transferring anelectron is generally a substance referred to as “electron carrier”,“mediator” or “oxidation/reduction(redox)-mediating agent”, such as theelectron transfer and the redox-mediating agent listed inJP-W-2002-526759.

In the biosensor, an inexpensive potassium ferricyanide (potassiumhexacyanoferrate (III)) is generally employed as an electron acceptor,and usually used at a final concentration of 1 mM or less. Nevertheless,the inventive coenzyme-binding glucose dehydrogenase enables a moresensitive measurement of D-glucose when using potassium ferricyanide ata concentration as high as 2 to 500 mM, more preferably 30 to 100 mM. Apreferred embodiment of the inventive measurement method, measurementreagent, measurement compound, biosensor and the like is characterizedby the use of potassium ferricyanide in its relevant measurement systemat a final concentration of 2 to 500 mM.

EXAMPLES

The present invention is further described in the following Examples, bywhich the invention is not restricted without departing from its scope.In the following Examples, the quantification of a coenzyme-bindingglucose dehydrogenase was conducted as described above.

Example 1 Culture of Deposited Strain 97508

100 ml of a culture medium (pH6.0) containing 1% glucose (WAKO PURECHEMICAL), 2% defatted soybean (NIPPON SHOKUHAN), 0.5% of corn steepliquor (KYODO SHOJI) and 0.1% magnesium sulfate (Nacalai Tesque) wasplaced in a 500-ml culture flask, which was sterilized at 121° C. for 20minutes, cooled, inoculated with a platinum loop of the deposited strain97508, shaken at 30° C. for 88 hours to obtain a seed culture of thestrain. 4 L of the culture medium having the composition similar to thatdescribed above but supplemented with an antifoam agent was added to a5-L jar fermenter, which was sterilized at 121° C. for 30 minutes,cooled, inoculated with 40 ml of the seed culture described above,cultured at 28° C. for 31 hours with aerating and shaking to obtain apreliminary culture of the strain. Then, 160 L of the culture mediumhaving the composition similar to that described above but supplementedwith an antifoam agent was added to a 200-L jar fermenter, which wassterilized at 121° C. for 20 minutes, cooled, inoculated with 1.6 L ofthe preliminary culture described above, cultured at 28° C. for 41 hourswith aerating and shaking. After completion of the culture, the culturefluid was centrifuged to obtain the supernatant.

Example 2 Isolation of Coenzyme-Binding Glucose Dehydrogenase from theCulture Supernatant

By the following Steps 2.1 to 2.5, the coenzyme-binding glucosedehydrogenase was isolated.

2.1 Concentration

160 L of the culture supernatant of Example 1 was concentrated throughan ultrafiltration membrane “Pellicon 2 Module” (Millipore), andtransferred into a 20 mM potassium phosphate buffer (pH7.5) to obtain acrude enzyme solution.

2.2 Purification by Butyl-TOYOPEARL 650M (TOSOH) (First Process)

The abovementioned crude enzyme solution was prepared in 65%-saturatedammonium sulfate (pH7.5), and centrifuged to obtain a supernatant. Thistreated crude enzyme solution was loaded onto a Butyl-TOYOPEARL 650Mcolumn (diameter: 4.7 cm, height: 7.7 cm) which had previously beenequilibrated with a 20 mM potassium phosphate buffer (pH7.5) containing65% ammonium sulfate; and thereby allowing the enzyme is absorbedtherein. This column was washed with the same buffer solution, and thenthe enzyme was allowed to be eluted with 20 mM potassium phosphatebuffer (pH7.5) containing 30% ammonium sulfate to collect an activefraction. The enzyme was further eluted by a gradient elution startingfrom the same buffer to 20 mM potassium phosphate buffer (pH7.5), andpooled with the former active fraction.

2.3. Purification by DEAE-CELLULOFINE A-500 (SEIKAGAKU KOGYO)

The abovementioned active fraction was concentrated through anultrafiltration membrane “Pellicon 2 Module”, desalted, and equilibratedwith a 15 mM Tris-HCl buffer (pH8.5). This fraction was loaded onto aDEAE-CELLULOFINE A-500 column (diameter: 4.7 cm, height: 5.2 cm) whichhad previously been equilibrated with the same buffer solution, and theeluate was collected.

2.4 Purification by Butyl-TOYOPEARL 650M (TOSOH) (Second Process)

The abovementioned eluate was prepared in 65%-saturated ammonium sulfate(pH7.5), and centrifuged to obtain a supernatant. This supernatant wasloaded onto a Butyl-TOYOPEARL 650M column (diameter: 4.7 cm, height: 3.6cm) which had previously been equilibrated with a 20 mM potassiumphosphate buffer (pH7.5) containing 65% ammonium sulfate; therebyallowing the enzyme is absorbed. This column was washed with the samebuffer solution, and then the enzyme was allowed to be eluted with 20 mMpotassium phosphate buffer (pH7.5) containing 30% ammonium sulfate tocollect an active fraction.

2.5 Purification by TSKgel G3000SW (TOSOH)

The abovementioned active fraction was concentrated through a penciltype membrane concentration module “ACP-0013” (ASAHI KASEI), desalted,and equilibrated with 50 mM potassium phosphate buffer (pH5.5)containing 0.2M sodium chloride. This fraction was loaded onto a TSKgelG3000SW (diameter: 2.15 cm, height: 60 cm) which had previously beenequilibrated with the buffer described above, and the enzyme was elutedwith the same buffer to obtain an active fraction. The active fractionwas concentrated through a centriplus 10 (Amicon), desalted, andtransferred into a 50 mM citric acid-sodium phosphate buffer (pH5.5).The resultant enzyme approximately had a specific activity of 1,100unit/mg, and a purification degree of about 170 times greater than thatof the crude enzyme solution.

Example 3 Test of Characteristics of Coenzyme-Binding GlucoseDehydrogenase

The coenzyme-binding glucose dehydrogenase isolated in Example 2described above was examined for its effect, optimum pH, pH forstability, optimum temperature, thermal stability, substratespecificity, molecular weight, inhibitor and coenzyme.

3.1 Effect

The coenzyme-binding glucose dehydrogenase was reacted with 500 mMD-glucose in the presence of 8.66 mM DCIP, and the reaction product wasquantified using D-gluconic acid/D-glucono-8-lactone measurement kit. Asa result, the production of D-gluconic acid was identified, and it wasrevealed that the inventive coenzyme-binding glucose dehydrogenase isthe enzyme that catalyzes a reaction for oxidizing a hydroxyl group inthe I-position of D-glucose.

3.2 Optimum pH

The buffer solution according to the Enzymatic activity measurementmethod 2 was replaced with the citric acid-sodium phosphate buffer(pH4.0 to 5.5), potassium phosphate buffer (pH6.5 to 7.5), Tris-HClbuffer (pH8.0 to 9.0) or glycine-sodium hydroxide buffer (pH9.5 to 10.0)(each 17 mM as a final concentration), and the enzymatic activity of thepurified enzyme was measured at various pH ranges similarly to Enzymaticactivity measurement method 2 (FIG. 1). As a result, the optimum pH ofthe coenzyme-binding glucose dehydrogenase was 7.0 to 9.0.

3.3 pH for Stability

The coenzyme-binding glucose dehydrogenase was dissolved in each 50 mMbuffer, i.e., citric acid-sodium phosphate buffer (pH3.2 to 6.4),potassium phosphate buffer (pH6.3 to 6.9), Tris-HCl buffer (pH7.3 to8.6) or glycine-sodium hydroxide buffer (pH9.1 to 11.4) and kept at 40°C. for 60 minutes, and then the enzymatic activity was examined by themethod according to the activity measurement method 1, and a ratio ofresidual enzymatic activity was analyzed (FIG. 2). As a result, the pHfor the stability of the coenzyme-binding glucose dehydrogenase waspH4.5 to 8.5.

3.4 Optimum Temperature

The coenzyme-binding glucose dehydrogenase was dissolved in 50 mM citricacid-sodium phosphate buffer (pH5.5) and examined for the enzymaticactivity over the range from 30° C. to 62° C. by the Enzymatic activitymeasurement method 1 described above (FIG. 3). As a result, the optimumtemperature of the coenzyme-binding glucose dehydrogenase was about 55°C.

3.5 Thermal Stability

The coenzyme-binding glucose dehydrogenase was dissolved in 50 mM citricacid-sodium phosphate buffer (pH5.5), kept at several points of thetemperature ranging from 0° C. to 55° C. for 15 minutes, and thenexamined for the enzymatic activity by the Enzymatic activitymeasurement method 1, and the ratio (%) of residual enzymatic activitywas analyzed (FIG. 4). The ratio (%) of residual enzymatic activity wascalculated with regarding the enzymatic activity after keeping at 0° C.for 15 minutes as 100%. As a result, the coenzyme-binding glucosedehydrogenase kept its enzymatic activity at a level as high as 89% evenat 50° C., showing the stability at about 50° C. or below.

3.6 Substrate Specificity and Km Value

Employing each of D-glucose and other substrates (each at 333 mM as afinal concentration, except for D-cellobiose at 193 mM, D-trehalose andD-raffinose at 121 mM), the enzymatic activity of this enzyme wasmeasured by the Enzymatic activity measurement method 1. The activity oneach substrate is represented as a relative value to the activity ofthis enzyme on D-glucose being regarded as 100%, and shown in Table 1.

Similarly, the relative reactivities (enzymatic activity) on D-glucoseand maltose each at two final concentrations, i.e. 550 mM and 100 mM,were measured. The results are represented as relative values based onthe value on D-glucose.

As evident from these results, the coenzyme-binding glucosedehydrogenase acts potently on D-glucose, and weakly on D-mannose,1,5-anhydro-D-glucitol, D-cellobiose, D-trehalose, maltose, D-galactose,D-glucose-6-phosphate and D-fructose. This enzyme exhibited almost noeffect on L-arabinose, lactose, D-sorbitol, gluconic acid, sucrose,D-mannitol, L-sorbose, D-ribose, L-rhamnose, D-glucose-1-phosphate,D-raffinose, ethanol or glycerol. The Km value of this enzyme was 49.7mM on D-glucose.

TABLE 1 Substrate Relative activity (%) D-Glucose 100 2-Deoxy-D-glucose48 D-Xylose 9.1 D-Mannose 2.8 1,5-Anhydro-D-glucitol 2 D-Cellobiose 2D-Trehalose 1.7 Maltose 1.4 D-Galactose 1.2 D-Glucose-6-phosphate 1.1D-Fructose 0.86 L-Arabinose 0.1> Lactose 0.1> D-Sorbitol 0.1> Gluconicacid 0.1> Sucrose 0.1> D-Mannitol 0.1> L-Sorbose 0.1> D-Ribose 0.1>L-Rhamnose 0.1> D-Glucose-1-phosphate 0.1> D-Raffinose 0.1> Ethanol 0.1>Glycerol 0.1>

TABLE 2 Substrate Final Conc.(mM) Relative activity (%) D-Glucose 550100 Maltose 550 2.8 D-Glucose 100 100 Maltose 100 0.53.7 Molecular Weight and Subunit Molecular Weight

The coenzyme-binding glucose dehydrogenase was dissolved in a 50 mMpotassium phosphate buffer (pH7.5) containing 0.2M NaCl, and analyzed ona TSKgel-G3000SW (diameter: 0.75 cm, length: 60 cm, TOSOH) using thesame buffer solution as a mobile phase. When using a molecular weightmarker (Oriental Yeast) as an index, the molecular weight of thecoenzyme-binding glucose dehydrogenase was revealed to be about 130 kDa.Using a 12.5% polyacrylamide gel, the inventive coenzyme-binding glucosedehydrogenase was subjected to an SDS-polyacrylamide gel electrophoresis(SDS-PAGE) according to the method by Laemmli et al (Nature, (1970)227:680-685). After the running, the gel was stained with Coomassiebrilliant blue, and the mobility was compared with that of the molecularweight marker (Amersham Pharmacia Biotech), which revealed that thesubunit molecular weight of the inventive coenzyme-binding glucosedehydrogenase was about 85 kDa.

3.8 Inhibitor

Each of the various additives shown in Table 3 was added as an inhibitorat 1 mM as a final concentration to the reaction system of the Enzymaticactivity measurement method 1, and the activity of the coenzyme-bindingglucose dehydrogenase was measured by the Enzymatic activity measurementmethod 1. In a control group, the procedure similar to that of Enzymaticactivity measurement method 1 was conducted except for adding noadditives shown in Table 3. Based on the enzymatic activity observed inthe control group being regarded as 100%, the activity in the presenceof each additive was calculated as a relative activity, the differenceof which from the control group activity was regarded as a % inhibitoryeffect. As a result, the inhibitory effect shown in Table 3 wasobserved.

On the other hand, in the Enzymatic activity measurement method 1described above, 1,10-phenanthroline respectively dissolved in methanolat 1 mM, 5 mM, 10 mM, 25 mM and 50 mM as final concentrations was added,and the activity of the inventive coenzyme-binding glucose dehydrogenasewas measured in accordance with the Enzymatic activity measurementmethod 1. The final concentration of methanol relative to each reactionsystem was 10% (v/v). In a control group, methanol was added in theEnzymatic activity measurement method 1 at 10% (v/v) as a finalconcentration. The results are shown in Table 4. It was revealed thatthe inhibitory effect of 1,10-phenanthroline was as high as 62.0% at 1mM, 76% at 5 mM, 85% at 10 mM, 91% at 25 mM and 95% at 50 mM as finalconcentrations of 1,10-phenanthroline.

The inhibitory effect on the inventive coenzyme-binding glucosedehydrogenase varied depending on the type of the additive, and was thehighest in the presence of heavy metal ion (such as Ag⁺, Cu²⁺ and Hg²⁺),and was 60% or more in the presence of 1,10-phenanthroline, proflavinand Mn²⁺.

TABLE 3 Additive Inhibition (%) None 0 NaN₃ 0 ZnCl₂ 0 AlCl₃ 0 Benzoicacid 0 EDTA 0.4 CdCl₂ 0.8 LiCl 0.9 Aminoguanidine sulfate 1.1 H₂O₂ 1.7N-Ethylmaleimide 1.8 Urea 1.9 NaCl 2.5 Tirone 2.5 BaCl₂ 2.6 PbCl₂ 2.7MgCl₂ 2.8 Fumaric acid 3.4 Cycloserine 3.6 DL-Penicillamine 4.3Meso-tartaric acid 5.6 Citric acid 5.6 CaCl₂ 5.7 Quinacrine 5.0TritonX-100 6.2 CoCl₂ 7.0 Malic acid 8.1 D-Tartaric acid 8.5 Iodoaceticacid 9.5 Cysteamine 9.8 2,2′-Bipyridine 10.8 8-Quinolinol 13.9 KCN 14.5NiCl₂ 16.5 FeCl₃ 25.0 Maleic acid 26.2 Acrinol 29.0 2-Nitrobenzoic acid44.3 SnCl₂ 45.5 Acriflavine 49.0 1,10-Phenanthroline 62.0 Proflavin 62.0MnCl₂ 75.5 AgNO₃ 99.4 CuCl₂ 100 HgCl₂ 100

TABLE 4 Final concentration of 1,10-phenanthroline (mM) Inhibit (%) 0 050 95 25 91 10 85 5 763.9. Coenzyme

The inventive coenzyme-binding glucose dehydrogenase solution wassupplemented with D-glucose and subjected to the absorption analysis,which indicated the disappearance of the maximum absorptions observed at385 nm and 465 nm in response to the supplement, revealing that thecoenzyme was flavin adenine dinucleotide. These maximum absorptions arespecific to FAD, and it was not observed in a control group reactionsystem constructed by excluding FAD only.

Example 4 Glucose Quantification

The coenzyme-binding glucose dehydrogenase derived from the depositedstrain 97508 purified in Example 2 described above was employed, and theabsorption reduction rate was measured using D-glucose at theconcentration ranging from 0.333 to 33 mM instead of 333 mM D-glucose inthe Enzymatic activity measurement method 1. Thus, the transition in theabsorption was measured at each D-glucose concentration of 0.333 mM, 0.5mM, 1 mM, 2 mM, 5 mM, 6.67 mM, 10 mM, 20 mM and 33 mM. The results ofthe measurement are shown in FIG. 5.

As a result, a calibration curve (correlation coefficient r=0.997) wasobtained; thereby, it was apparent that the quantification of D-glucoseemploying the coenzyme-binding glucose dehydrogenase is possible.

Example 5 Measurement of Glucose by Enzyme-Immobilizing Electrode

The coenzyme-binding glucose dehydrogenase derived from the depositedstrain 97508 purified in Example 2 described above was employed tomeasure D-glucose by an enzyme-immobilizing electrode. A glassy carbon(GC) electrode on which 3.4 U of this enzyme was immobilized wasemployed to measure the response current to the glucose concentration.In an electrolytic cell, 2.7 ml of 100 mM sodium phosphate buffer(pH7.0) and 0.3 ml of 1M aqueous solution of potassium hexacyanoferrate(III) (potassium ferricyanide) were added. The GC electrode wasconnected to the potentiostat BAS100B/W (BAS), and the solution wasstirred at 40° C. in each condition of argon saturation, oxygensaturation and air saturation while applying +500 mV to thesilver-silver chloride reference electrode. 30 μl of a 1M D-glucosesolution was added to these systems, and the current value in thestationary phase was measured. The same amount of the 1M D-glucosesolution was further added, and the current value was measured, theseprocedure being repeated each three times. The resultant current valuesvs the known glucose concentrations (about 10, 20, 30 and 40 mM) wereplotted to obtain a calibration curve (FIG. 6). Thereby, it was apparentthat the quantification of glucose by the enzyme-immobilizing electrodeemploying the coenzyme-binding glucose dehydrogenase is possible. It wasalso revealed, based on the consistent calibration curve obtainedregardless of any gas saturated conditions, that the coenzyme-bindingglucose dehydrogenase is extremely inert to the oxygen and that it ispossible to quantify D-glucose by the enzyme-immobilizing electrodeutilizing the enzyme without being subjected to any effect of theoxygen.

Example 6 Measurement of Glucose in Standard Serum byEnzyme-Immobilizing Electrode

Analogous to Example 5, the concentration of glucose in a serum wasmeasured by the enzyme-immobilizing electrode using the control serum IWAKO B (WAKO PURE CHEMICAL). In an electrolytic cell, 2.4 ml of 100 mMsodium phosphate buffer (pH7.0) and 0.3 ml of 1M aqueous solution ofpotassium hexacyanoferrate (III) (potassium ferricyanide) were added,and the serum was added when the current value became stationary and thecurrent value was measured. Similarly, the D-glucose solutions of knownconcentrations were measured to obtain a calibration curve. The glucoseconcentration in the serum was identified by the calibration curvemethod to be 4.5 mM, which was in agreement with the concentration ofthe glucose identified by the hexokinase-glucose-6-phosphatedehydrogenase method. Accordingly, it was revealed that thequantification of D-glucose in serum by the enzyme-immobilizingelectrode employing the coenzyme-binding glucose dehydrogenase employedin the invention is possible.

INDUSTRIAL APPLICABILITY

According to the present invention, it became possible to provide asoluble coenzyme-binding glucose dehydrogenase whose activity on altoseis 5% or less and which is inhibited by 1,10-phenanthroline.Furthermore, a method for producing the coenzyme-binding glucosedehydrogenase suitable to an industrial production and a microorganismproducing therefor are also provided. As a result, it becomes possibleto apply the coenzyme-binding glucose dehydrogenase to an industrialapplication, and more particularly, it becomes possible to measure theblood sugar level even in a diabetes patient receiving an infusioncontaining maltose. Also by using the inventive coenzyme-binding glucosedehydrogenase, a trace amount of the glucose can be measured even with aglucose sensor, thus enabling the utility. It also becomes possible touse in a material production or analysis including a method formeasuring or eliminating glucose in a sample using the coenzyme-bindingglucose dehydrogenase as well as a method for producing an organiccompound, thereby providing a highly utilizable enzyme, which enablesuse for modifying a material in the fields of pharmaceuticals, clinicalstudies and food products.

The invention claimed is:
 1. A method for producing a biosensor formeasuring glucose in a sample liquid comprising: (i) obtaining a solubleflavin adenine dinucleotide-binding glucose dehydrogenase (FAD-GDH)secreted from an Aspergillus fungal body, which has enzymatic activityto glucose comprising catalyzing a reaction for oxidizing glucose in thepresence of an electron acceptor, and (ii) forming a biosensorcomprising an electrode system and an enzymatic reaction layer on anelectrode of the electrode system, the enzymatic reaction layercomprising the soluble flavin adenine dinucleotide-binding glucosedehydrogenase and an electron acceptor, wherein: (a) enzymatic activityof the FAD-GDH to maltose is 5% or less relative to the enzymaticactivity of the FAD-GDH to glucose, and (b) enzymatic activity of theFAD-GDH to D-fructose is not more than enzymatic activity of the FAD-GDHto D-mannose.
 2. The method for producing a biosensor of claim 1,wherein the enzymatic activity to maltose is 3% or less relative to theenzymatic activity to glucose.